The objectives of this research program are to elucidate the pathway of ubiquinone biosynthesis in eukaryotic cells with emphasis on (a) the identity of the intermediates, (b) the nature of the enzymes involved in each step, including the mechanism of the reaction, and (c) the factors which regulate the pathway under various conditions. It has been demonstrated that ubiquinone-9 biosynthesis occurs in rat liver mitochondria and the ubiquinone-6 biosynthesis can occur in yeast mitochondria. Two new intermediates in ubiquinone-6 biosynthesis have been discovered in mutants of S. cerevisiae, namely 3,4-dihydroxy-5-hexaprenylbenzoate and 3-methoxy-4-hydroxy-5-hexaprenylbenzoate. These findings indicate that yeast, and presumably eukaryotic cells, have an alternate pathway for converting 4-hydroxy-5-polyprenylbenzoate to 6-methoxy-2-polyprenylphenol. It has also been found that one of these intermediates, 3,4-dihydroxy-5-hexaprenylbenzoate acid accumulates during catabolite repression of yeast cells with exogenous glucose. Studies will be carried out to determine the mechanism of this metabolite repression using inhibitors of protein synthesis and selected effectors for enzymatic regulation. The regulatory enzyme (SAM-3,4-dihydroxy-5-polyprenylbenzoate methyl transferase) will be purified and its properties studied.